RU2360671C2 - Application of alpha-ketoglutaric acid for treatment of undernutrition or condition with high-glucose plasma level - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to pharmacology. The method for improving amino acid absorption in a vertebrate, including a mammal and a bird, includes introduction to a vertebrate of AKG (alpha-ketoglutaric acid), mono- and dimetallic AKG salts, chitosan-AKG or their mixtures in amount and/or at rate sufficient to maintain the desired effect. The method for decreasing plasma glucose absorption in a vertebrate, including a mammal and a bird, involves introduction to a vertebrate, including a mammal and a bird, of AKG, mono- and dimetallic AKG salts, chitosan-AKG or their mixtures in amount and/or at rate sufficient to maintain the desired effect of glucose absorption. The method of prevention, inhibition or relief of the condition with high-glucose plasma level in a vertebrate, including a mammal and a bird includes introduction to a vertebrate, including a mammal and a bird, of AKG, mono- and dimetallic AKG salts, chitosan-AKG or their mixtures in amount and/or at rate sufficient to maintain the desired effect on the specified condition.

EFFECT: application of AKG, mono- and dimetallic AKG salts, chitosan-AKG or their mixtures in therapeutically effective amount for making the composition used to prevent, relieve and treat the condition with high-glucose plasma level, application of AKG, mono- and dimetallic AKG salts, chitosan-AKG or their mixtures for making the composition used to improve absorption, changed absorption, deteriorated absorption and disordered absorption of amino acids and/or peptides.

19 cl, 3 tbl, 3 ex, 1 dwg

Description

FIELD OF THE INVENTION

This invention relates to a method for improving the absorption of amino acids, as well as to a method for reducing the absorption of glucose in a vertebrate animal, including a mammal and a bird. Also contemplated is the manufacture of a composition for improving the absorption of amino acids in said vertebrate.

BACKGROUND OF THE INVENTION

Diabetes mellitus is a serious metabolic disease characterized by the presence of constantly elevated plasma glucose levels. The classic symptoms of diabetes in adults are polyuria, polydipsia, acetonuria, rapid weight loss combined with elevated plasma glucose levels.

Normal fasting plasma glucose concentrations are less than 115 milligrams per deciliter. In patients with diabetes, fasting plasma glucose concentrations above 140 milligrams per deciliter are detected. As a rule, diabetes develops in response to damage to the pancreatic beta cells. This damage can be caused by primary diabetes mellitus, in which beta cells are destroyed by the autoimmune system, or by a secondary diabetic response to other primary diseases, such as pancreatic disease, hormonal disorders, except for the absence of insulin action, drug or chemical induction, insulin receptor abnormalities , genetic syndromes, etc.

Primary diabetes mellitus can be classified as type I diabetes (also called insulin-dependent diabetes mellitus or IDDM) or type II diabetes mellitus (also called non-insulin-dependent diabetes mellitus or NIDDM).

Type I diabetes, juvenile or insulin-dependent diabetes, is a well-known hormone-deficient condition in which pancreatic beta cells are destroyed by the body's own immune defenses. In patients with type I diabetes, the ability to secrete endogenous insulin is weak or absent. These patients develop severe hyperglycemia. Type I diabetes was fatal until the introduction of insulin replacement therapy about 70 years ago — first using insulin from animal sources, and most recently using human insulin made using recombinant DNA technology. It is now clear that the destruction of beta cells in type I diabetes leads to a combined deficiency of two hormones, insulin and amylin. When pancreatic cells are destroyed, the ability to secrete insulin and amylin is lost.

The nature of pancreatic beta cell damage in type II diabetes is unclear. Unlike pancreatic beta cells of type I diabetics, type II beta cells of diabetics retain the ability to synthesize and secrete insulin and amylin. Type II diabetes is characterized by insulin resistance, that is, insufficiency of the normal metabolic response of peripheral tissues to the action of insulin. In other words, insulin resistance is a condition in which circulating insulin generates an insufficient biological response. In clinical terms, insulin resistance is present when normal or elevated plasma glucose levels persist against normal or elevated insulin levels. Hyperglycemia associated with type II diabetes can sometimes be reversed or weakened with a diet or weight reduction sufficient to restore peripheral tissue sensitivity to insulin. In fact, type II diabetes is often characterized by hyperglycemia in the presence of elevated, compared with normal, plasma insulin levels. The progression of type II diabetes is associated with increased plasma glucose concentrations and is associated with a relative decrease in glucose-induced insulin secretion rate. For example, in the late stage of type II diabetes mellitus, insulin deficiency may be present.

Known treatment and prevention of diabetes

The primary goal in the treatment of all forms of diabetes is the same, namely: reducing plasma glucose concentrations to values as close to normal as possible, and thereby minimizing both short-term and long-term complications of this disease (Tchobroutsky, Diabetologia 15: 143-152 (1978)).

The relationship between the degree of hyperglycemia in diabetes and the resulting long-term complications was further confirmed in the recently completed Clinical Test for Diabetes and its Complications (DCCT, Diabetes Control and Complications Trial) undertaken by The Diabetes Control and Complications Trial Research Group, N Eng. J. Med. 329: 977 (1993)). DCCT was conducted over a 10-year period at 29 clinical centers throughout the United States and Canada and showed that lower average plasma glucose concentrations in type I diabetes reduced receptor complications. The development of retinopathy decreased by 76%, the progression of retinopathy by 54%, the signs of renal disease (proteinuria, albuminuria) also decreased. Significant neuropathic changes were also reduced.

In the treatment of type I diabetes, parenteral administration of replacement doses of insulin is inevitably involved. In combination with a proper diet and self-monitoring of plasma glucose, most type I diabetics can achieve a certain level of plasma glucose control.

In contrast to type I diabetes, treatment for type II diabetes often does not require insulin. The therapeutic treatment system for type II diabetes usually includes diet therapy and lifestyle changes, usually usually within 6-12 weeks.

Features of the diabetic diet include adequate, but not excessive, total calorie intake, regular meals, restriction of saturated fat, concomitant increase in the content of polyunsaturated fatty acids and increased consumption of dietary fiber.

Changes in lifestyle include maintaining regular physical activity, which contributes to both the regulation of weight and a decrease in the degree of resistance to insulin.

If fasting hyperglycemia persists after an adequate diet and lifestyle changes, then a diagnosis of “primary malnutrition” can be made, and then, for the regulation of plasma glucose and thereby minimizing the complications of the disease, either oral hypoglycemic therapy or the insulin therapy system itself will be needed. Type II diabetes, which does not respond to diet and weight loss, may respond to therapy with oral hypoglycemic agents, such as sulfonylureas or biguanides. Insulin therapy, however, is used to treat other patients with type II diabetes, especially those who have failed in the primary diet and are not obese, or those who have failed in both the primary diet and secondary oral hypoglycemic therapy.

The use of amylin agonists in the treatment of diabetes is described in US Pat. Nos. 5,124,314 and 5,175,145. Excess amylin activity mimics the main symptoms of type II diabetes, and amylin blockade has been proposed as a new therapeutic strategy.

Well-known therapeutic agents are, for example, diabetic pills based, for example, on sulfonylureas, which help the pancreas produce more insulin and help the body make better use of insulin. Possible side effects: hypoglycemia, upset stomach, skin rash or itching and weight gain.

Other pills are based on biguanides, which limit the production of glucose by the liver, as well as reduce the amount of insulin in the body, improve blood fat and cholesterol. Possible side effects include a painful condition combined with alcohol, worsening existing kidney problems, weakness, dizziness, shortness of breath, nausea, and diarrhea.

Other pills are based on alpha glucosidase inhibitors and block enzymes that break down starch. Possible side effects are stomach problems.

Other pills are based on thiazolidinediones, which help cells become more sensitive to insulin. Possible side effects are that they should not be used with concomitant liver disease (regular checks), hypoglycemia and use only in combination with other therapy, as well as the less effective effect of birth control pills, weight gain, the risk of anemia, swelling (edema).

Other pills are based on meglitinides, which help the pancreas produce more insulin after eating. Possible side effects are hypoglycemia and weight gain.

In addition, there is a combination of oral medications based, for example, on glyburide (sulfonylurease) and metformin (biguanide), under the name, for example, "Glucovance". Possible side effects are hypoglycemia, the inability to use for kidney disease, and the undesirability of use in combination with alcohol.

US Pat. No. 5,234,906 discloses compositions containing glucagon and an amylin agonist, and their use for the regulation or treatment of hyperglycemic conditions.

WO 93/10146 discloses amylin agonists and their use for the treatment or prevention of hyperglycemic conditions, including insulin-dependent conditions such as diabetes mellitus.

Renal failure and malnutrition

Renal failure or renal dysfunction is a condition in which the kidneys are not able to purify the blood from waste. Renal failure causes an accumulation of toxic waste in the blood. The kidneys normally have excess cleansing ability, and kidney function can be 50% of normal before symptoms appear. Symptoms include itching, fatigue, nausea, vomiting, loss of appetite, leading to malnutrition. Renal failure is often associated with diabetes and high blood pressure. The symptoms mentioned above, that is, vomiting and loss of appetite, lead to malnutrition in a subject suffering from renal failure.

Dialysis reduces the impact of waste on the kidneys. However, this procedure takes a lot of time, and the patient may need it several times a week. A patient undergoing a dialysis procedure needs medical supervision, and this procedure is both expensive and time consuming.

Glutamate oxidation

Thanks to in situ studies in rats Windmueller and Spaeth (1), it is known that glutamate and glutamine are an important metabolic fuel for the small intestine. Windmueller and Spaeth were the first to report a significant partial metabolism of glutamate (≈95%) and glutamine (≈70%) in the gastrointestinal tract during absorption. These results have since been confirmed in vivo in both piglets (2) and humans (3).

In the process of glutamate oxidation, the first stage is transamination with any number of enzymes, deamination with glutamate dehydrogenases (GDH), many of which are expressed in the gastrointestinal tract (4, 5). Deamination with GDH leads to the formation of AKG (alpha-ketoglutaric acid) and free ammonia. In the process of transamination with branched chain aminotransferase (BCAT), glutamate transfers the amino group to a branched α-keto acid to form AKG and the corresponding branched amino acid.

Alpha Ketoglutaric Acid

Glutamine and its derivatives, for example alpha-ketoglutaric acid (AKG), are molecules that play a central role in systemic and intestinal metabolism through the Krebs cycle. However, the mechanisms are still not fully understood (Pierzynowski, SG and Sjödin, A. (1998) J. Anim. A. Feed Sci. 7: 79-91; and Pierzynowski, SG et al. Eds: KBK Knutsen and JE Lindberg, Uppsala June 19-21, 2001).

AKG (2-oxo-pentanedioic acid, 2-oxoglutaric acid, alpha-oxoglutaric acid, alpha-oxopentanedioic acid, 2-ketoglutaric acid, 2-oxo-1,5-pentanedioic acid, 2-oxopentanedioic acid, 2-oxoglutaric acid) theoretically, it can be a product of the breakdown of glutamine, glutamate, glutamic acid during the metabolism in the body. It can also serve as a precursor not only for glutamine and arginine, but also for some other amino acids, and therefore it is considered as a catabolic protein protector. Olin et al., 1992, showed that when AKG was added to fish feed, urine output decreased. Similarly, in humans, when AKG is added to solutions for total parenteral nutrition (TPN) mixed with other amino acids, there is good protection against nitrogen loss after surgery (Pierzynowski, SG and Sjödin, A. (1998) J. Anim. A. Feed Sci 7: 79-91). In the case of humans, AKG probably combines with the breakdown of muscle proteins to serve the needs of the intestinal tract during so-called postoperative stress, such as catabolism, starvation, etc.

In Riedel E. et al., Nephron 1996, 74: 261-265, which is the closest analogue of the present invention, it is shown that administration of α-ketoglutarate with calcium carbonate effectively improves amino acid metabolism in hemodialysis patients.

The need for metabolites from the glutamine family for intestinal functioning has recently been proven by Reeds et al. (1996, Am. J. of Physiol. - Endocrinology and Metabolism 270: 413-418), which reported an almost 100% utilization of glutamate / glutamine upon first passage through the small intestine of piglets.

AKG can be an important energy donor through several conversion pathways, for example via ornithine and putrescine to GABA (gamma-aminobutyric acid) or succinate. Theoretically, AKG can also act as an acceptor of an ammonium ion, possibly through conversion to glutamate / glutamine.

It is known - but never published - that enterocytes during their growth depend on ammonium.

Therefore, in light of the aforementioned problems, it is highly desirable to develop means and methods of treating and preventing hyperglycemic conditions, such as diabetes mellitus, as well as malnutrition, often associated with diabetes and, for example, renal failure, in mammals, for example cats, dogs or humans, in which the problems or side effects associated with the means and methods of the prior art could be avoided. There is also a need to improve well-being in addition to nutritional status in both renal and diabetic patients. In this regard, the present invention is directed to these needs and interests.

SUMMARY OF THE INVENTION

From the point of view of the above disadvantages, known in the field of preventing, treating and / or alleviating diabetes, as well as other related hyperglycemic diseases, and the high cost of medical care at the same time, as well as for correcting nutritional deficiencies associated, for example, with diabetes and renal failure, the present invention provides new and improved methods and compositions for preventing, treating and / or alleviating diabetes and malnutrition.

An object of the present invention is to provide a method for improving the absorption of amino acids in a vertebrate animal, including a mammal and a bird. This method comprises administering to a vertebrate animal, including a mammal and a bird, AKG, derivatives or metabolites of AKG, AKG analogs, or a mixture thereof, in an amount and / or frequency sufficient to provide the desired effect on amino acid absorption.

In one embodiment of this method, AKG, AKG derivatives or metabolites, AKG analogues or mixtures thereof are selected from the group consisting of alpha-ketoglutaric acid (AKG), ornithine-AKG, arginine-AKG, glutamine-AKG, glutamate-AKG, leucine-AKG , chitosan-AKG and other salts of AKG with amino acids and amino acid derivatives; mono- and dimetallic salts of AKG, such as CaAKG, Ca (AKG) ₂ and NaAKG.

In yet another embodiment, the vertebrate is a rodent such as a mouse, rat, guinea pig or rabbit; a bird such as a turkey, chicken, chicken or other broilers; farm animals such as a cow, horse, pig, piglet or other freely moving farm animals; or a pet such as a dog or cat.

In another embodiment, the vertebrate is a human.

In yet another embodiment, the amino acid is any essential amino acid.

In a further embodiment, the essential amino acid is isoleucine, leucine, lysine and proline.

In addition, the invention includes a method of reducing glucose absorption in a vertebrate animal, including a mammal and a bird. This method involves administering to the vertebral, including mammalian and poultry, AKG, derivatives or metabolites of AKG, AKG analogues or mixtures thereof in an amount and / or frequency sufficient to provide the desired effect on glucose absorption.

In addition, the invention includes a method for preventing, inhibiting, or alleviating a high glucose condition in a vertebrate, including a mammal and a bird. This method comprises administering to a vertebrate animal, including a mammal and a bird, AKG, derivatives or metabolites of AKG, AKG analogs or mixtures thereof in an amount and / or frequency sufficient to provide the desired effect with respect to said condition.

In one embodiment, the high glucose condition is type I or type II diabetes mellitus.

The invention further includes the use of AKG, derivatives or metabolites of AKG, AKG analogues or mixtures thereof, for the manufacture of a composition for the prevention, amelioration or treatment of a high glucose condition.

In one embodiment, the high plasma glucose condition is type I or type II diabetes mellitus.

The invention also relates to the use of AKG, derivatives or metabolites of AKG, AKG analogues or mixtures thereof for the manufacture of a composition for the prevention, alleviation or treatment of malnutrition.

In one embodiment of use, the composition is a pharmaceutical composition, optionally with a pharmaceutically acceptable carrier and / or additives.

In another embodiment, the use of the composition is a food or nutritional supplement.

In yet another embodiment, the food or nutritional supplement is a dietary supplement and / or component in the form of a solid food and / or drink.

In yet another embodiment, AKG, derivatives or metabolites of AKG, AKG analogs, or mixtures thereof, are formulated in a therapeutically effective amount.

In yet another embodiment, the therapeutically effective amount is 0.01-0.2 g / kg body weight per daily dose.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

The drawing shows the kinetics of leucine in the whole body in control and AKG infused pigs. Values are mean ± SEM (standard error); n = 9, each pig received both control and AKG. Values for AKG did not differ from control using analysis of variance (ANOVA). AKG - α-ketoglutarate; NOLD - non-oxidative removal of leucine; Ra is the rate of leucine appearance; Balance - Ra, subtracted from NOLD, is a protein residue in the body by leucine.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

In the context of this application and invention, the following definitions are used.

The term "pharmaceutical composition", as used here, refers to a therapeutically effective composition according to the invention.

The term “therapeutically effective amount”, or “effective amount” or “therapeutically effective”, as used herein, refers to such an amount that provides a therapeutic effect for a given condition and mode of administration. This is a predefined amount of the active substance, calculated so as to produce the desired therapeutic effect in combination with the necessary additive and diluent, that is, a carrier or diluent for administration. In addition, this term is intended to mean an amount sufficient to reduce, and most preferably prevent, a clinically significant deficiency in the activity, function and response of the host. Alternatively, the therapeutically effective amount is sufficient to cause an improvement in the clinically significant condition in the host. It will be apparent to those skilled in the art that the amount of compound may vary depending on its specific activity. Suitable dosages may contain a predetermined amount of the active composition, calculated so as to produce the desired therapeutic effect in combination with the necessary diluent, that is, a carrier or additive. In methods and applications for the manufacture of the compositions of the invention, a therapeutically effective amount of an active component is provided. A therapeutically effective amount can be determined by a mid-level medical or veterinarian based on patient characteristics such as age, weight, gender, condition, complications, other diseases, etc., as is well known in the art.

The term "derivative" in this description is intended to mean a chemical substance obtained from the starting material either directly or by modification or partial substitution.

The term "analogue" in this description is intended to refer to compounds that are structurally similar to others, but are not necessarily isomers. Analogs have similar function (s), but differ in structure or evolutionary origin.

As used herein, the term “treatment” refers to treatment for the purpose of cure, which may be a complete / final or partial cure of a condition or conditions.

The term "facilitate" in this description is intended to mean not only a decrease in the intensity of the condition or symptom, but also a delayed onset of the condition or symptom.

The term “warn” in this description is intended to mean a guarantee that an event does not happen, for example, a condition or symptom related to an underdeveloped GIT (gastrointestinal tract) does not occur. As a result of preventing a particular condition or symptom, the onset of such a condition or symptom is delayed.

The term "increased absorption of amino acids" in this description is intended to indicate a change in the overall absorption of amino acids in a vertebrate compared with a vertebrate not receiving treatment or administration according to the invention. Changes are considered as an increase if the overall absorption is quantitatively higher in the specified vertebrate compared with the vertebrate of the same species not receiving the specified treatment.

The term "kinetics" in this description is intended to mean constant or frequent monitoring or measurement of absorption of amino acids, as well as glucose in a vertebrate animal to determine the rate of absorption.

The term “sodium AKG,” as used herein, is used interchangeably with the terms AKG-Na, Na-AKG, Na-salt AKG, AKG (Na-salt).

The term “chitosan-AKG”, as used herein, is used interchangeably with the terms “AKG-chitosan”, “AKG (chitosan salt)”.

Diagnosis of type I and type II diabetes

The diagnosis of patients with type I and type II diabetes is within the qualifications of specialists in this field of technology. For example, individuals over the age of 35 with symptoms of polydipsia, polyuria, polyphagia (with or without weight loss) in combination with elevated plasma glucose concentrations and no history of ketoacidosis are usually considered as part of the diagnosis of type II diabetes mellitus. The presence of obesity, a positive family history of type II diabetes and normal or elevated fasting plasma insulin and c-peptide concentrations are additional characteristics of most patients with type II diabetes mellitus. By “therapeutically effective amount” is meant an amount which, either in single or multiple doses, favorably reduces plasma glucose concentrations in a subject affected by type II diabetes mellitus.

Now, the inventors have unexpectedly discovered that the infusion site has an effect on the absorption of AKG. After duodenal infusion of AKG, an increased absorption of amino acids and a decreased absorption of glucose were unexpectedly observed.

Therefore, the present invention can be used to reduce plasma glucose in a subject with type II diabetes mellitus.

Diagnosis of malnutrition

The diagnosis of patients with malnutrition, that is, with poor or insufficient nutrition or hypotrophy, is within the qualifications of a specialist in this field of technology. Typically, an assessment of an individual’s overall health is performed to assess malnutrition.

Diagnosis of renal failure

The diagnosis of patients affected by renal failure, is within the qualifications of a specialist in this field of technology.

There are two forms of renal failure, acute and chronic renal failure (ACF and CRF). Acute renal failure can usually be reversed, while chronic renal failure usually progresses. CRF treatment is divided into predialysis and active treatment of uremia using, for example, dialysis or transplantation. There is no exact definition of predialysis as a starting point, but predialysis is usually defined as the period of time between the diagnosis of renal failure and the start of active treatment. Dialysis and transplantation are considered as an active treatment.

A method for improving the absorption of amino acids

According to the invention, a method for improving the absorption of amino acids in a vertebrate, including a mammal and a bird, is disclosed. This method comprises administering to a vertebrate, including a mammal and a bird, AKG, derivatives or metabolites of AKG, AKG analogs, or a mixture thereof, in an amount and / or frequency sufficient to provide the desired effect on amino acid absorption.

The absorption of amino acids is considered to be improved when compared with the absorption of amino acids in a vertebrate animal, including a mammal and a bird that does not receive these AKGs, AKG derivatives or metabolites, AKG analogues or mixtures thereof.

In further embodiments of this method, AKG, AKG derivatives or metabolites, AKG analogues or mixtures thereof are selected from the group consisting of alpha-ketoglutaric acid (AKG), ornithine-AKG, arginine-AKG, glutamine-AKG, glutamate-AKG, leucine-AKG , chitosan-AKG and other salts of AKG with amino acids and amino acid derivatives; mono- and dimetallic salts of AKG, such as CaAKG, Ca (AKG) ₂ and NaAKG.

In further embodiments, the vertebrate is a rodent, such as a mouse, rat, guinea pig or rabbit; a bird such as a turkey, chicken, chicken or other broilers; farm animals such as a cow, horse, pig, piglet or other freely moving farm animals; or a pet such as a dog or cat.

In a further embodiment, the vertebrate is a human. A person may be a patient in need of treatment for malnutrition due to, for example, kidney failure, diabetes mellitus, sports, age (children and the elderly), pregnancy, anorexia nervosa, bulimia nervosa, Bing's eating disorder, compulsive overeating, or other non-specific eating disorders ( EDNOS).

A vertebrate animal, such as the specified person, in the following embodiments can be any vertebrate that needs to increase the availability and utilization of amino acids, for example essential amino acids or conditionally essential amino acids, in particular isoleucine, leucine, lysine and proline.

Examples of essential amino acids are alpha amino acids such as isoleucine (IIeu), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), threonine (Thr), tryptophan (Try) and valine (Val) people. Essential amino acids vary between species. Rats need two other amino acids, namely arginine (Arg) and histidine (His).

The following embodiments are those where the amino acid is any amino acid such as alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine, threonine, cysteine, tyrosine, glutamine, histidine, lysine, arginine, aspartate, asparagine, glutamate, glutamine, glycine and serine.

The following embodiments are those where the amino acid is any essential or conditionally essential amino acid. Examples of essential or conditionally essential amino acids are shown in table 2.

In a further embodiment, essential or conditionally essential amino acids are selected from the group consisting of isoleucine, leucine, lysine and proline.

A method for reducing glucose absorption and a method for preventing, inhibiting or alleviating an increase in plasma glucose

Plasma glucose is the amount of glucose (sugar) in the blood. It is also known as serum glucose. The amount of glucose in the blood is expressed in millimoles per liter (mmol / L) or in mg / dl.

Normally, plasma glucose levels in humans throughout the day remain within narrow limits, from about 4 to 8 mmol / L. Plasma glucose levels are higher after meals and are usually the lowest in the morning. Fasting glucose levels are normally about 70-110 mg / dl (3.9-6.1 mmol / l), and 2 hours after eating, normal levels are about 80-140 mg / dl (4.4-7, 8 mmol / l). Plasma glucose> 180 mg / dl (> 10.0 mmol / L) 2 hours after a meal is usually considered high plasma glucose. This is also true for plasma glucose> 140 mg / dL on an empty stomach.

If a person has, for example, diabetes, his plasma glucose level sometimes shifts beyond these limits. The main disadvantage in all patients with diabetes is the reduced ability of insulin to induce the removal of glucose (sugar) molecules from the blood cells of the body. Regardless of whether this reduced insulin activity is a consequence of a reduced amount of insulin produced (e.g. type I diabetes) or insensitivity of cells to a normal amount of insulin, the result is the same, i.e. too high plasma glucose levels. This is called "hyperglycemia," which means "high blood glucose concentration." Hyperglycemia is usually diagnosed when plasma glucose is more than 240 mg / dL (> 13.4 mmol / L).

According to the invention, a method for reducing plasma glucose absorption in a vertebrate animal, including a mammal and a bird, is disclosed. This method comprises administering to the vertebral, including mammalian and poultry, AKG, derivatives or metabolites of AKG, AKG analogues or a mixture thereof in an amount and / or frequency sufficient to provide the desired effect on glucose absorption.

The decrease in glucose absorption after administration of AKG, AKG derivatives or metabolites, AKG analogues or mixtures thereof can be 5-50%, for example 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50% of the initial glucose value in plasma.

In a further embodiment, the reduction in absorption is 20-40% of the initial plasma glucose value.

In a further embodiment, the decrease is 30% of the initial plasma glucose value.

In addition, a method for preventing, inhibiting or alleviating a condition with a high plasma glucose concentration in a vertebrate animal, including a mammal and a bird, is disclosed. The method comprises administering to the vertebral, including mammalian and poultry, AKG, derivatives or metabolites of AKG, AKG analogues or mixtures thereof in an amount and / or frequency sufficient to provide the desired effect with respect to the indicated condition with a high plasma glucose concentration.

In a further embodiment, the high plasma glucose state is a hyperglycemic state.

These methods related to conditions with a high concentration of plasma glucose or to hyperglycemic conditions include the following embodiments, where AKG, derivatives or metabolites of AKG, AKG analogues or mixtures thereof are selected from the group consisting of alpha-ketoglutaric acid (AKG), ornithine- AKG, arginine-AKG, glutamine-AKG, glutamate-AKG, leucine-AKG, chitosan-AKG and other AKG salts with amino acids and amino acid derivatives; mono- and dimetallic salts of AKG, such as CaAKG, Ca (AKG) ₂ and NaAKG.

Further, further embodiments are those wherein the vertebrate is a rodent, such as a mouse, rat, guinea pig or rabbit; a bird such as a turkey, chicken, chicken or other broilers; farm animals such as a cow, horse, pig, piglet or other freely moving farm animals; or a pet such as a dog or cat.

In addition, the following embodiments are those where the vertebrate is a human.

In addition, in the following embodiments, these conditions with a high plasma glucose concentration are the result of, for example, acromegaly, Cushing's syndrome, hyperthyroidism, pancreatic cancer, pancreatitis, pheochromocytoma, insufficient insulin, or excessive food intake.

In addition, in the following embodiments, these high plasma glucose conditions are a consequence of type I or type II diabetes mellitus.

The use of AKG for diabetes and for the treatment of malnutrition

The invention discloses the use of AKG, derivatives or metabolites of AKG, AKG analogues or mixtures thereof, for the manufacture of a composition for the prevention, alleviation or treatment of a condition with a high plasma glucose concentration.

Examples of conditions with a high concentration of plasma glucose and hyperglycemic conditions are given in the previous paragraph.

The following embodiments include those in which the hyperglycemic state is type I or II diabetes mellitus.

The invention discloses the use of AKG, derivatives or metabolites of AKG, AKG analogues or mixtures thereof for the manufacture of a composition for the prevention, alleviation or treatment of malnutrition.

In further embodiments of these applications, said composition is a pharmaceutical composition optionally with a pharmaceutically acceptable carrier and / or additives.

In further embodiments, the composition is a food or nutritional supplement.

In further embodiments, the food or nutritional supplement is a dietary supplement and / or component in the form of a solid food and / or drink.

In further embodiments, AKG, derivatives or metabolites of AKG, AKG analogues, or mixtures thereof, are formulated in a therapeutically effective amount.

In further embodiments, the therapeutically effective amount is 0.01-0.2 g / kg body weight per daily dose.

Introduction AKG, derivatives or metabolites of AKG, analogues of AKG or mixtures thereof

According to the methods disclosed above, AKG, AKG derivatives or metabolites, AKG analogues or mixtures thereof are administered to a vertebrate, including a mammal and a bird; a rodent such as a mouse, rat, guinea pig or rabbit; a bird such as a turkey, chicken, chicken or other broilers; farm animals such as a cow, horse, pig, piglet or other freely moving farm animals; or a pet such as a dog or cat.

The introduction can be carried out in different ways, depending on what type of vertebrate is to be treated, on the condition of the vertebral animal in need of such methods, and on the specific indication for treatment.

In one embodiment, the administration is in the form of a food or food supplement, such as a dietary supplement and / or a component in the form of a solid food and / or drink. The following embodiments may be in the form of suspensions or solutions, such as the beverage described below.

Also, dosage forms may include capsules or tablets, such as chewable or soluble, such as effervescent tablets, as well as powder and other dry forms known to those skilled in the art, such as pills, such as micropills, granules and grains.

Administration may occur in the form of parenteral, rectal or oral nutrition or a dietary supplement, as shown above.

Parenteral carriers include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, Ringer's solution with lactate, or fatty oils.

A food and nutritional supplement may also be emulsified. The active therapeutic ingredient can then be mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerin, ethanol or the like, and combinations thereof. In addition, if desired, the composition may contain small amounts of auxiliary substances, such as moisturizing or emulsifying agents, pH-, buffering agents, which enhance the effectiveness of the active ingredient.

Various forms of parenteral food or nutritional supplements can be proposed, such as solid foods, liquids, or lyophilic or otherwise dried formulations. They may include diluents from various buffers (e.g. Tris-HCl, acetate, phosphate), for pH and ionic strength, additives such as albumin or gelatin, to prevent absorption on surfaces, detergents (e.g. Tween 20, Tween 80, Pluronic F68, bile salts), solubilizing agents (e.g. glycerin, polyethylene glycerin), antioxidants (e.g. ascorbic acid, sodium metabisulfite), preservatives (e.g. thimerosal, benzyl alcohol, parabens), bulk substances or tonicity modifiers (e.g. lactose, mannitol), forged attaching polymers, such as polyethylene glycol, to the composition, complexing with metal ions, or incorporating a substance inside or onto the surface of granular preparations of polymer compounds such as polyacrylic acid, polyglycolic acid, hydrogels, etc. or liposomes, microemulsions, micelles, monolayer or multilayer vesicles, erythrocyte shadows or spheroplasts.

Drink

In one embodiment, the food or nutritional supplement is administered in the form of a beverage or a dry composition thereof by any of the methods of the invention.

The drink contains an effective amount of AKG, AKG derivatives or metabolites, AKG analogues or mixtures thereof, together with a nutritionally acceptable water-soluble carrier such as minerals, vitamins, carbohydrates, fat and proteins. Examples of AKG, derivatives or metabolites of AKG, analogues of AKG or mixtures thereof are alpha-ketogluaric acid (AKG), ornithine-AKG, arginine-AKG, glutamine-AKG, glutamate-AKG, leucine-AKG, chitosan-AKG and other AKG salts with amino acids and amino acid derivatives; mono- and dimetallic salts of AKG, such as CaAKG, Ca (AKG) ₂ and NaAKG.

All of these components are supplied in dry form if the beverage is supplied in dry form. The drink, supplied ready to drink, additionally contains water. The finished beverage solution may also have adjustable tonicity and acidity, for example as a buffer solution according to the general sentences in the above paragraph.

The pH is preferably in the range of about 2-5, and in particular about 2-4, to prevent the growth of bacteria and fungi. You can also use a sterilized drink with a pH of about 6-8.

The drink may be supplied separately or in combination with one or more therapeutically effective compositions.

Use of AKG, derivatives or metabolites of AKG, AKG analogues or mixtures thereof

The invention discloses the use of AKG, AKG derivatives or metabolites, AKG analogues or mixtures thereof for the manufacture of a composition for the prevention, alleviation or treatment of hyperglycemic conditions such as type I and type II diabetes, as well as for the treatment of malnutrition.

The following embodiments of the invention include use, wherein the composition is a pharmaceutical composition. This pharmaceutical composition may be together with a pharmaceutically acceptable carrier and / or additives, such as diluents, preservatives, solubilizing agents, emulsifying agents, adjuvants and / or carriers, useful in the methods and applications disclosed in the present invention.

In addition, when used here, "pharmaceutically acceptable carriers" are well known to those skilled in the art and may include, but are not limited to, 0.01-0.05 M phosphate buffer or 0.8% saline. In addition, such pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcohol / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral carriers include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, Ringer's solution with lactate, or fatty oils. Preservatives and other additives may also be present, such as, for example, antimicrobial agents, antioxidants, chelating agents, inert gases and the like.

The following embodiments of the invention include use, wherein the composition is a dietary supplement and / or component in the form of a solid food and / or drink.

Such a formulated composition, for example a pharmaceutical composition, or a food or food supplement, contains the composition of the invention and may further comprise a carrier and / or some amount of a second or additional active ingredient affecting any hyperglycemic condition, such as type I and II diabetes, as well as malnutrition.

Dose of the administered pharmaceutical composition

According to the invention, the use of AKG, derivatives or metabolites of AKG, AKG analogues or mixtures thereof for the manufacture of a composition of the invention comprises the administration of a therapeutically effective amount to a vertebrate animal such as a bird or mammal in need thereof. Such a therapeutically effective amount is about 0.01-0.2 g / kg body weight per daily dose.

AKG, derivatives or metabolites of AKG, AKG analogues or mixtures thereof

According to the invention, AKG, derivatives or metabolites of AKG, AKG analogues or mixtures thereof are included. Examples of AKG, AKG derivatives or metabolites, AKG analogues or mixtures thereof are alpha-ketoglutaric acid (AKG), ornithine-AKG, arginine-AKG, glutamine-AKG, glutamate-AKG, leucine-AKG, chitosan-AKG and other AKG salts with amino acids and amino acid derivatives; mono- and dimetallic salts of AKG, such as CaAKG, Ca (AKG) ₂ and NaAKG.

Targets for the introduction

As one of ordinary skill in the art can readily understand, the methods and pharmaceutical compositions of the present invention are particularly suitable for administration to any vertebrate animal in need thereof, such as poultry, including turkey, chicken or chicken, and other broilers, but not limited to, and freely moving animals, or mammals, including domestic animals, such as, but not limited to, feline or canine, farm animals, such as representatives of cows, horses, goats, sheep and pigs, but not limited to them, wild animals, either in nature or in the zoological garden, experimental animals such as mice, rats, rabbits, goats, sheep, pigs, dogs, cats, etc., i.e. for veterinary use .

People are also included as targets for administering in the treatment of any high plasma glucose or hyperglycemic state, such as type I and type II diabetes, as well as any condition associated with malnutrition, after, for example, renal failure, type I diabetes and type II.

In addition, any vertebrate animals, such as those mentioned above, that need to increase the availability and utilization of amino acids, for example essential amino acids or conditionally essential amino acids, in particular isoleucine, leucine, lysine and proline, can also be targets for administration. A person may also be a patient in need of treatment of malnutrition or to increase the availability and utilization of amino acids due to, for example, renal failure, surgical interventions, such as pancreatectomy or transplantation, geriatric conditions, diabetes mellitus, exercise, age (children and elderly people), pregnancy, anorexia nervosa, bulimia nervosa, Bing's eating disorder, compulsive overeating, eating disorders, metabolic disorders or other non-specific disorders in nutrition (EDNOS), bedsores, lack of appetite in a vertebrate animal or due to debilitating illness.

References

(All references cited here are incorporated into this description by reference in full)

1. Windmueller, H. G., & Spaeth, A. E. (1975) Intestinal metabolism of glutamine and glutamate from the lumen as compared to glutamine from blood, Arch. Biochem. Biophys. 171: 662-672.

2. Stoll B., Bun-in, D. G., Henry, J, Hung, Y, Jahoor, F, & Reeds, P. J. (1999) Substrate oxidation by the portal drained viscera of fed piglets. Am. J. Physiol. 277: E168-E175.

3. Matthews, D. E., Marano, M. A., & Campbell, R. G. (1993) Splanchnic bed utilization of glutamine and glutamic acid in humans. Am. J. Physiol. 264: E848-E854.

4. Madej, M., Lundh, T., & Lindberg J. E. (1999) Activities of enzymes involved in glutamine metabolism in connection with energy production in the gastrointestinal tract epithelium of newborn, suckling and weaned piglets. Biol. Neonate 75: 250-258.

5. Suryawan, A., Hawes, J. W., Hards, R.A., Shimomura, Y., Jenkins, A. E., & Hutsun, S. M. M. (1998) A molecular model of human branched-chain amino acid metabolism. Am. J. Clin. Nutr. 68: 72-81.

6. Lambert, B. D., Stoll, B., Niinikoski, H., Pierzynowski, S., & Bun-in, D.G. (2002) Net portal absorption of enterally fed alpha-ketoglutarate is limited in young pigs. J. Nutr. 132: 3383-3386.

7. Kristensen, N. B., Jungvid, H., Femandez, J. A., & Pierzynowski, S. G. (2002) Absorption and metabolism of a-ketoglutarate in growing pigs. J. Anim. Physiol. Anim. Nutr. 86: 239-245.

8. Bergmeyer, H. U., & Bemt, E. (1974) 2-oxoglutarate. UV spectrophotometric determination. In: Methods of enzymatic analysis, 2nd Ed. (Bergmeyer, H. U., ed.). Academic Press, New York, NY.

9. Pajor, A. M. (1999) Sodium-coupled transporters for krebs cycle intermediates. Annu. Rev. Physiol. 61: 663-682.

10. Murphy, J. M., Murch, J. M., and Ball, R. O. (1996) Proline is synthesized from glutamate during intragastric infusion but not during intravenous infusion in neonatal piglets. J. Nutr. 126: 878-886.

EXAMPLES

The invention is illustrated below by a number of non-limiting examples.

Although the invention has been described with respect to the specific embodiments disclosed, one skilled in the art may anticipate other embodiments, variations, or combinations that are not specifically mentioned, but nevertheless fall within the scope of the appended claims.

Section Materials and Methods for Examples 1-2

Keeping animals

Keeping and caring for animals was in line with US Department of Agriculture guidelines.

Study design

Female piglets (n = 9) were purchased from the Texas Department of Criminal Justice, Huntsville, TX.

Piglets (14 days old) were brought to the Children's Nutrition Research Center and kept on a diet of liquid milk replacer (Litter Life, Merrick, Middleton, WI) at a rate of 50 g / (kg · day) for a 7-day adaptation period.

The composition of the milk replacer (per kg dry matter) was 500 g of lactose, 100 g of fat and 250 g of protein.

After 7 days during the night, the piglets were left without food and prepared for surgery, as described previously (2).

Describing briefly, under isoflurane anesthesia under aseptic conditions, the pigs were implanted with a polyethylene catheter (external diameter 1.27 mm, Becton Dickinson, Sparks, MD) in the common portal vein and sylastic catheters (external diameter 1.78 mm) in the jugular external vein and carotid artery .

An ultrasonic flow sensor (internal diameter from 8 to 10 mm, Transonic, Ifhaca, NY) was placed around the portal vein.

A silicone catheter (2.17 mm outer diameter, Baxter Healthcare, McGaw Park, IL) was implanted into the duodenum. The catheters were filled with sterile saline containing heparin (2.5 × 10 ⁴ U / L) and brought out either on the left side (vessels of the portal and duodenal catheters, flow sensors), or between the shoulder blades (jugular catheter and carotid artery catheter).

Immediately prior to surgery, animals received an intramuscular injection of an antibiotic (20 mg / kg enrofloxacin, Bayer, Shawnee Mission, KS) and an intramuscular injection of an analgesic (0.1 mg / mg butorphenol tartrate. Fort Dodge Labs, Fort Dodge, IA).

Before resuming enteral nutrition after surgery, the piglets were kept on completely parenteral nutrition for 24 hours at a rate of 5 ml · kg ^-1 · h ^-1 . Piglets were given 7 days to recover from surgery. In all piglets, the absorption of food and the rate of weight gain returned to preoperative levels.

Sample preparation

Blood samples were immediately placed on ice and centrifuged.

Plasma was collected, immediately frozen in liquid N ₂ and stored at -80 ° C until analysis.

Amino Acid Analysis

For amino acid analysis of the plasma, a 0.2 ml aliquot of the plasma was mixed with an equal volume of an aqueous solution of methionine sulfone (4 mmol / L) and centrifuged at 10,000 × g for 120 min through a 10 kDa cut-off filter.

A 50 μl aliquot of the filtrate was dried and amino acids were analyzed by reverse phase HPLC of their phenylisothiocyanate derivatives (Pico Tag, Waters, Wobum, MA).

Plasma AKG was determined by the method of Bergmeyer and Bemt (8) with minor modifications.

Briefly described, the analysis was performed in 0.5 ml of a working solution consisting of 100 mmol / L phosphate buffer (pH 7.6), 4 mmol / L ammonium chloride and 50 μmol / L NADH.

An appropriate amount of plasma containing 1-10 nmol AKG was added to the working solution.

The initial absorption was obtained at 340 nm.

After recording the initial absorbance, ~ 6 units (in a volume of 10 μl) of bovine GDH (G2501; Sigma-Aldrich, St. Louis, MO) were added to each tube.

After a 10-minute incubation, a second absorption reading was taken at 340 nm.

The amount of AKG in the sample is directly proportional to the decrease in absorption between the first and second readings.

The concentration of AKG was calculated using a standard curve.

Determination of ammonia in plasma

Plasma ammonia was determined using a spectrophotometric assay kit (171-C, Sigma-Aldrich, St. Louis, MO).

Plasma glucose determination

Plasma glucose was determined using a spectrophotometric assay kit (315-100; Sigma-Aldrich, St. Louis, MO).

Determination of bicarbonate in the blood

To evaluate blood enrichment with bicarbonate, an aliquot of whole blood (1.0 ml) was placed in 10 ml of a Vacutainer (Becton Dickinson, Franklin Lakes, NJ) and 0.5 ml of perchloric acid (10% w / w) was added.

Room air (10 ml) filtered through soda lime (Sodasorb; Grace Container Products, Lexington, MA) was injected into a Vacutainer, withdrawn into a gas-tight syringe and transferred to a second Vacutainer.

The carbon dioxide isotope enrichment in the gas sample was measured on a continuous mass flow spectrometer to determine the isotope ratio (ANCA; Europa Instruments, Crewe, U.K.).

Determination of ketoisocaproic acid in plasma

Plasma ketoisocaproic acid (KIC) was isolated by cation exchange chromatography (resin AG-50V, Bio-Rad).

The eluents were treated with sodium hydroxide (100 μl; 10 N) and hydroxylamine HCl (200 μl; 0.36 M) and heated (60 ° C; 30 min). After cooling, the pH of the samples was adjusted to a value of <2.

Keto acids were extracted into 5 ml of ethyl acetate and dried under nitrogen at room temperature.

Derivation of KIC was carried out by adding 50 μl of a mixture of N-methyl-N-tert-butyl-dimethylsilyl-trifluoroacetamide + 1% tert-butyl-dimethylchlorosilane.

KIC isotope enrichment was determined by El GC-MS (gas chromatography - electron impact ionization mass spectrometry (Hewlett Packard 5970 GC mass spectrometer with Hewlett Packard 5890 Series II GC) by monitoring ions at 316 m / z and 317 m / z .

Determination of isotopic enrichment of plasma urea

Plasma urea isotopic enrichment was determined by El GC-MC analysis. Proteins were precipitated from 50 μl of plasma with 200 μl of ice-cold acetone.

After shaking, the protein was separated by centrifugation, and the supernatant was collected and dried in a nitrogen atmosphere.

To the dried supernatant was added 250 μl of 1:20 diluted bis (dimethylacetal) malonic aldehyde and concentrated HCl (30 wt%), the sample was incubated at room temperature for 2 hours, and then evaporated to dryness (Speedvac, Savant Instruments, Forma Scientific Marietta, OH).

Urea derivatives were prepared using 50 μl of a mixture of N-methyl-N-tert-butyl-dimethylsilyl-trifluoroacetamide + 1% tert-butyl-dimethylchlorosilane, and plasma isotope enrichment was determined using El GS-MS analysis by monitoring ions with m / z 153-155.

Calculations

The net residue of metabolites in the portal vein [μmol / (kg · h)] was calculated as follows:

where is Conc. represents blood concentration (μmol / L), PORT and ART refer to portal vein blood and arterial blood, and PBF represents portal vein blood flow [L / (kg · h)].

The flow of leucine in the whole body [Q; μmol / (kg · h)] was calculated as follows:

where R represents the rate of infusion of the labeled atom [μmol / (kg · h)] and

IE _infusate and IE _plasma are isotopic enrichments (expressed in mol%) of the infused labeled atom and plasma KIC, respectively.

The production of CO ₂ in the body was calculated as follows:

where IE _infusate is the enrichment of H ¹³ CO ₃ ^- in the infusate (excess molar percentage), IE _{arterial bicarbonate} is the enrichment in arterial blood (excess molar percent) and the rate of infusion of tracer [mol / (kg h)] during intravenous bicarbonate infusion, which was continued at each treatment period. The entire equation was divided by 0.82 to correct for the compensation of the infused labeled carbon in bicarbonate.

The oxidation of leucine in the whole body [μmol / (kg · h)] was calculated as follows:

представляет собой изотопное обогащение бикарбоната во время инфузии 1-С¹³-лейцина и IE_LEU представляет собой изотопное обогащение 1-C¹³-KIC во время инфузии 1-С¹³-лейцина.Where

is an isotopic enrichment of bicarbonate during infusion 1 ¹³ C-leucine and IE _LEU is an isotopic enrichment of 1-C ¹³ -KIC during infusion 1 ¹³ C-leucine.

The non-oxidative removal of leucine in the whole body (NOLD) is an assessment of the incorporation of leucine into the muscles. NOLD [μmol / (kg · h)] was calculated using the following equation:

The rate of appearance of leucine in the whole body (Ra) [μmol / (kg · h)] is an estimate of protein catabolism, and it was calculated as:

The urea flow in the whole body was calculated as follows:

where IE represents the enrichment of the infusate, PE represents the enrichment of plasma in a stable state during urea infusion, and IR represents the infusion rate.

Statistical analysis

For all statistical criteria, p = 0.05 was considered to be of statistical significance.

In Example 1, the effect of AKG on the kinetics, appearance in the arteries, in the portal vein and the net appearance in the portal vein of individual amino acids, AKG, glucose, ammonia and leucine were analyzed using the general linear model method (Minitab. Inc., State College, PA). This model included the effects of adding AKG and a pig. The pig was included as a random variable. The test average was calculated on a computer using the LSMEANS function. Student's one-sided t-test was used to check whether the net AKG residue in the portal vein was significantly higher than zero during the control treatments.

Example 1 - Measurement of AKG, glucose, plasma ammonia, blood flow and urea flow in the whole body

goal

The purpose of this example is to evaluate the effect of AKG infusion on AKG, glucose, plasma ammonia, blood flow and the urea flow in the whole body.

Animal experiments

Piglets were deprived of food for 15 hours before the start of the experiment.

On the day of the experiment, at time 1 hour, with a primary dose (7.75 ml / kg; 25% w / w aqueous solution; oral) continuous duodenal infusion of milk replacer [Litter Life, Merrick, Middleton, WI; 7.75 ml / (kg · h)] was prepared as a 25% (w / w) aqueous solution, which provided ~ 920 kJ and 12.5 g of protein / (kg · day).

Either saline (control; 930 mmol / L NaCl) or sodium AKG (Na-AKG) 930 mmol / L from Sigma-Aldrich, St. Louis, MO was dissolved in a milk replacer.

The level of AKG was selected based on previous data (6) from the laboratory, when more than 2.5% of the dry matter of food was required to observe the AKG residue found in the portal vein.

Piglets also received an intravenous (200 μmol / kg) continuous 6-hour infusion of ¹⁵ N ₂ urea [20 μmol / (kg · h)] (98%; Cambridge Isotope Laboratories).

At time 0 h, a continuous 2-hour infusion of NaH ¹³ CO ₂ (15 μmol / (kg · h); 99%; Cambridge Isotope Laboratories, Andover, MA) was started with a primary dose (15 μmol / kg).

Arterial samples were obtained 0, 90, 105, and 120 minutes after the start of the NaH ¹³ CO ₂ infusion to determine CO ₂ production in the whole body.

At a time of 2 hours, the NaH ¹³ CO ₂ infusion was stopped and started with an initial dose (40 μmol / kg) of a continuous 4-hour infusion of 1- ¹³ C-leucine (40 μmol / (kg · h); 99%; Cambridge Isotope Laboratories) .

Arterial and portal vein samples were obtained at time 4, 5, and 6 h to determine the kinetics of leucine and urea, as well as the mass balance of ammonia, AKG, glucose, and amino acids.

All pigs received both control and AKG treatment in a fully randomized fashion with at least 24 hours intervals between treatment periods.

results

AKG, glucose, plasma ammonia, blood flow and urea flow in the whole body are presented in table 1.

Table 1. The effect of AKG infusion on the concentration of metabolites, the net residue in the portal vein and the kinetics of 1- ¹³ C-leucine
and ¹⁵ N ₂ urea in the whole body. AKG ¹ (% dry matter of food) 0 3.75 R AKG infusion rate μmol / (kg · h) 0 930 - Portal blood flow, l / (kg · h) 3.21 ± 0.28 ² 3.36 ± 0.27 0.34 Arterial AKG, µmol / L 13.8 ± 1.7 27.4 ± 3.6 <0.01 AKG in the portal vein, μmol / L 22.0 ± 1.4 64.6 ± 5.9 <0.001 Net AKG residue in portal vein, µmol / (kg · h) 19.7 ± 2.8 95.2 ± 12 <0.001 Net balance AKG portal vein,% of infused - 10.23 ± 0.57 - The net residue of glucose in the portal vein, µmol / (kg · h) 303.1 ± 61 203.9 ± 69 <0.05 The net residue of ammonia in the portal vein, µmol / (kg · h) 520.1 ± 66 561.1 ± 53 0.91 Urea flow in the whole body, μmol / (kg · h) 398.3 ± 35 377.8 ± 39 0.56 ¹ AKG, α-ketoglutarate; ² SEM (standard deviation)

AKG infusion [930 μmol / (kg · h)] increased (P <0.01) the concentration of AKG in the arteries and portal vein and the net AKG residue in the portal vein. Even when AKG was not infused into the duodenum, net AKG absorption in the portal vein [19.7 ± 2.8 μmol / (kg · h)] was significantly higher than 0. However, net AKG absorption in the portal vein was increased (P <0.001) when processing AKG compared to control. The net AKG residue in the portal vein was 95 μmol / (kg · h), which is only 10.23% of the infused amount.

The net residual in the portal vein of 10.23% actually represents some reassessment of the absorption of the infused AKG, since when only saline was infused, a statistically significant absorption of AKG occurred. When adjusting for AKG absorption from a control food, the proportion of infused AKG that appears in the portal vein drainage is reduced to 8.12%.

Interestingly, the net glucose residue in the portal vein was reduced (P <0.05) during AKG treatment. AKG treatment did not affect the blood flow in the portal vein, the net ammonia residue in the portal vein, and the urea flow in the whole body.

Proline concentrations in both arteries and portal vein were increased (P <0.05), and leucine in the portal vein tended (P <0.01) to increase during AKG treatment (data not shown). The mass residue of amino acids in the portal vein is presented in Table 2. The AKG treatment increased (P <0.05) the mass residue of leucine, lysine and proline in the portal vein and tended to increase the mass residue of isoleucine (P <0.10).

Table 2. Pure amino acid residues in the portal vein in pigs receiving a duodenal infusion of 0 or 930 μmol / (kg · h) AKG (n = 5). Amino acid The control AKG ¹ The remainder in the portal vein The remainder in the portal vein μmol / (kg · h) % revenue μmol / (kg · h) % revenue Essential Amino Acids Isoleucine 164.5 ± 26 100.1 230.2 ^b ± 28 140.0 Leucine 294.9 ± 44 76.3 438.6 ^a ± 50 113.4 Phenylalanine 80.4 ± 11 83.3 95.2 ± 11 98.7 Valine 218.5 ± 33 85,2 279.3 ± 32 108.9 Histidine 27.7 ± 11 43.1 45.9 ± 3.8 71,4 Threonine 185.0 ± 40 66,4 210.9 ± 18 75.7 Lysine 237.7 ± 35 72.3 324.5 ^a ± 37 98.8 Tryptophan 38.6 ± 6.4 - 47.2 ± 4.3 - Conditionally Essential Amino Acids Arginine 95.2 ± 24 85.8 109.0 ± 19 98.3 Proline 216.4 ± 25 69.9 354.5 ^a ± 32 114.5 Tyrosine 85.7 ± 12 100.6 115.8 ± 17 135.9 Non-Essential Amino Acids Alanine 539.6 ± 61 182.9 557.8 ± 48 189.0 Aspartate 28.2 ± 4.6 9.2 29.7 ± 6.0 9.6 Asparagine 169.9 ± 23 - 185.6 ± 18 - Glutamate 64.2 ± 23 14.9 80.1 ± 17 18.6 Glutamine 17.2 ± 12 - 25.5 ± 45 - Glycine 167.0 ± 27 109,4 177.2 ± 20 116.0 Serine 213.3 ± 89 94.4 244.7 ± 64 108.3 ^a differs from the control (P≤0.05); ^b Differs from control (P <0.10) ¹ AKG, α-ketoglutarate; ² Mean ± SEM

The kinetics of leucine in the whole body is shown in the drawing. AKG treatment did not affect the body flux, NOLD, Ra, and oxidation.

Example 2 - Measurement of the average disappearance of AKG in the lumen

goal

The purpose of this example is to evaluate the average disappearance of an infused AKG bolus in the lumen.

Animal experiments

Pigs (n = 7) were given a duodenal bolus infusion (7.75 ml / kg; 25% (w / w) aqueous solution) of a liquid milk replacer (Litter Life, Merrick) containing 25 mg / ml sodium AKG (1040 μmol / kg BW).

After 1 h, the pigs were killed.

The small intestine was carefully clamped in the proximal part of the duodenum and the distal part of the ileum, and 2 x 50 ml of saline was removed and washed with a stream to rinse the intestines.

Washings were collected, combined, and an aliquot of 15 ml was instantly frozen in liquid N ₂ and stored at -80 ° C for subsequent AKG analysis.

results

AKG was infused with a bolus of 1040 μmol / kg. The average disappearance in the lumen was 663 ± 38 μmol / kg in one hour. This value represents 63.8 of 1040 μmol / kg infused AKG.

Discussion and general conclusion from experiment 1 and 2

In Example 1, AKG was continuously infused into the duodenum, and only 10% of the infused AKG appeared in the portal vein drainage.

The observation that only 10% of infused AKG appeared in portal vein plasma increases the likelihood of some variants of AKG fate in the lumen. One possible explanation for the small appearance of AKG in the portal vein is that AKG transport in the lumen is limited. Sodium / dicarboxylate co-transporters capable of transporting AKG exist on the membranes of the pig brush border (9), so it seems unlikely that AKG will not be captured by enterocytes. To verify this, the inventors infused one duodenal bolus of 1040 μmol / kg and found that more than 660 μmol / kg disappeared from the small intestine of the piglets in 1 h (Example 2). Thus, approximately 64% of the AKG bolus disappeared from the lumen of the duodenum in just 1 hour.

AKG infusion did not affect the net disappearance of glutamate and glutamine in the portal vein, as was previously observed (6). If the absorbed AKG was converted to glutamate, it would either be released into the portal vein blood or converted to other amino acids.

One would, however, expect that the release of glutamate and glutamine would not increase by AKG, even if a substantial conversion to these amino acids occurs, given that very little dietary glutamate or glutamine is released by PDV (portal drain viscera, the inside of the drained portal vein) in a normal diet (references 1, 2). It was shown (10) that proline can be synthesized from intestinal glutamate by intestinal tissue. Given that the increase in net proline residue in the portal vein was 138.1 μmol / (kg · h) in pigs treated with AKG, and that more than 800 μmol / (kg · h) AKG was not taken into account in the portal vein residue, it is possible that an increase in the net proline residue in the portal vein may be entirely the result of conversion from AKG. However, such a significant conversion of AKG to proline in the enterocyte would lead to a decrease in the ammonia residue in the portal vein, but the ammonia residue in the portal vein remained unchanged. The lack of effect on the ammonia residue in the portal vein was also reflected in similar rates of urea synthesis in the two groups.

Branched chain amino acid transaminase (BCAA) catalyzes the interaction between AKG and branched chain amino acids (leucine, isoleucine and valine). BCAAs are transaminated to form glutamate from AKG and the corresponding keto acid from each BCAA. Additional AKG can lead to a decrease in the net release of BCAAs from PDV by stimulating the transamination of BCAAs with the formation of glutamate. However, leucine release in the portal vein was increased by AKG, although this did not affect the kinetics of leucine in the whole body. The net lysine residue in the portal vein was also increased by AKG. Due to the fact that the net residue of many amino acids in the portal vein was about 100% for many amino acids during AKG treatment, it is not clear whether AKG saved amino acids or increased the release of amino acids due to proteolysis in the interior of the drained portal vein.

In addition, the probable fate of AKG inside the enterocyte is oxidation through the tricarboxylic acid cycle (TCA). If indeed all of the carbon infused as AKG was oxidized to CO ₂ , one would expect an increase in the yield of CO ₂ from PDV, while the production of CO ₂ in the whole organism does not increase with AKG infusion. Interestingly, the net glucose residue in the portal vein was reduced by AKG treatment.

Due to the fact that significant amounts of AKG disappeared from the lumen of the small intestine, but this cannot be explained by drainage of the portal vein in relation to AKG or in relation to the net balance of amino acid products of AKG metabolism, the fate of AKG during enteral nutrition remains unclear. However, when AKG was infused into the duodenum, only 10% of the lumen supply appeared in the portal vein drainage, although this amount of AKG was sufficient to increase the remainder in the portal vein and the concentration of this compound in the blood circulation. Thus, despite the uncertainty regarding the exact metabolic fate of AKG in the lumen, these results indicate that the availability of dietary AKG from the intestine is limited.

The resulting increase in AKG in the blood circulation did not have an effect on the net appearance of glutamate, glutamine, ammonia, and BCAAs in the portal vein.

In addition, increased systemic AKG had no effect on the kinetics of leucine in PDV or in the whole body or on urea flow. These results are consistent with previous data when AKG was delivered intragastrically.

Example 3 - Comparative effect of Na-AKG and chitosan-AKG, administered enterally, on the resorption of amino acids and keto acids in enterocytes and blood plasma and their metabolism

goal

The purpose of this example is to compare the effects of Na-AKG (or AKG Na-salt) and enteric chitosan-AKG on the resorption of amino acids and keto acids in enterocytes and blood plasma and their metabolism. The effects of Na-AKG and chitosan-AKG on the conversion of keto acids to amino acids were also measured by monitoring plasma levels of amino acids. This study tests the hypothesis that AKG affects the conversion of keto acids to amino acids in the intestines and improves protein synthesis.

Animal experiments

In this experiment, only three pigs were used; these pigs had a body weight of approximately 20 kg. Pigs were divided into cages and fed standard food for 4-5 days to adapt to new devices. Then, pigs were surgically implanted with catheters and intestinal cannulas and given 3-7 days for recovery.

Used surgical procedures were procedures commonly used in the art and known to those skilled in the art.

After the operation, in this case, a 3-day recovery period was provided, and pigs were fed once a day (at time 10:00) with standard food (3% of body weight). After the recovery period, the level of amino acids in the blood plasma was measured under the conditions of the introduction of Na-AKG (see experiment (2)), the introduction of chitosan-AKG (experiment (3)) and without the introduction of AKG (experiment (1); control experiment), additional details which are given below.

AKG administration conditions.

Experiment (1).

Keto acids or amino acids (Amines) (total volume 50 ml) were infused intraduodenally (id) at a dose of * "morning meal equivalent" for 1 hour.

10 portions were given in 1 h (50 ml dose + 50 ml saline).

This experiment was a control experiment.

(* "Morning feed equivalent" means that the animals received approximately the same amount of amino acids as is usually found in the feed corresponding to the morning meal).

Blood samples were taken (at baseline ** level, 0 h) and after 1, 2, 4 hours.

(** Baseline is determined as a sample at time 0 prior to the infusion of amino acids / keto acids.)

Blood samples (5 ml of whole blood for amino acid analysis from an artery, portal, hepatic vein) were collected in ethylenediaminetetraacetic acid (EDTA) with aprotinin to stop coagulation and proteinase activity.

(Processing may include the use of 5 drops of EDTA + trasylol, centrifugation and plasma freezing at -20 ° C.)

Experiment (2).

Keto acids or amino acids (Amines) mixed with Na-AKG (in a total volume of 50 ml) were infused intraduodenally (ID) at a dose of * "morning meal equivalent" for 1 hour (10 servings were given in 1 h, 50 ml doses, possibly with saline).

Blood samples were taken (at baseline, 0 h) and after 1, 2, 4 hours.

Blood samples (5 ml of whole blood for amino acid analysis from an artery, portal, hepatic vein) were collected in ethylenediaminetetraacetic acid (EDTA) with aprotinin to stop coagulation and proteinase activity.

Experiment (3).

Keto acids or amino acids (Amines) mixed with chitosan-AKG (in a total volume of 50 ml) were infused intraduodenally (etc.) at a dose of * "morning meal equivalent" for 1 hour (10 servings were given in 1 h, 50 ml doses, possibly with saline).

Blood samples were taken (at baseline, 0 h) and after 1, 2, 4 hours.

Blood samples (5 ml of whole blood for amino acid analysis from an artery, portal, hepatic vein) were collected in ethylenediaminetetraacetic acid (EDTA) with aprotinin to stop coagulation and proteinase activity.

results

Table 3 below shows the results of this study.

Table 3. Incremental increase in free amino acids in the blood after the introduction of amino acids Time (h) ΔI (mmol / l) ΔII (mmol / L) ΔI (mmol / l) ΔII (mmol / L) Artery Artery Hepatic Hepatic vein vein one -0.5 ^a 0.65 ⁱⁿ 0.43 ^A 0.23 ^A 1,5 0.04 ^b 1.1 ^g 1.6 ^B 1.85 ^BV 2.5 -0.48 ^a 1.69 ^g 1.59 ^B 1.94 ^V

I is a Na-AKG salt

II is a salt of chitosan-AKG

Δ-increment in time = (amino acids at Δ-time 0 - amino acid level after 1, 1.5 and 2.5 hours)

Distinguishing small or capital letters given with the results describe statistical differences at p <0.05.

Discussion and general conclusions to Example 3

This example shows that the chitosan-AKG salt improves the absorption of essential amino acids. This improvement is greater than that achieved with Na-AKG. This observation is important and essential for better utilization of dietary amino acids to improve the absorption of amino acids in the damaged intestinal tissue, found, for example, in diabetic patients or the elderly.

EXAMPLES OF FOOD (DIETARY) ADDITIVES AND / OR COMPONENTS

AKG, mono- and dimetallic salts of AKG or chitosan-AKG can be used as an active agent.

Drink

Composition of the drink (per 1000 liters):

Active agent 0.4% Sweetener 0.225% Lemon acid 0.5% Vanilla flavor 0.01% Vitamin C 0.05% Carbon dioxide 2.0% Water up to 100%

The drink is prepared using a standard method. The ingredients, with the exception of citric acid, ascorbic acid and carbon dioxide, are mixed in a suitable tank equipped with a mechanical stirrer. Then add citric acid and ascorbic acid and mix thoroughly for 15-20 minutes. Add the remaining water. The resulting mixture was saturated with carbon dioxide and poured into suitable containers.

Pet food

Feed Composition:

Active agent 0.4% Gelatin 4.8% Wheat bran 5.7% Vitamins and Minerals 7.7% Corn starch 25.4% Powdered milk 11.6% Casein 25.7% Yeast 8.7% Beef fat 9% Sunflower oil one%

The specified composition is prepared by simple mixing of these components in accordance with traditional technologies and packaged in standard packaging weighing 0.25, 0.5 and 1 kg.

Claims (19)

1. A method of improving the absorption of amino acids in a vertebrate animal, including a mammal and a bird, in which AKG (alpha-ketoglutaric acid), AKG mono- and dimetallic salts, chitosan-AKG, or mixtures thereof, are administered to a vertebrate, including a mammal and a bird, and / or with a frequency sufficient to provide the desired effect on the absorption of amino acids. 2. The method of claim 1, wherein the mono- and dimetallic salts of AKG are selected from the group consisting of CaAKG, Ca (AKG) _2, and NaAKG. 3. The method according to claim 1, where the vertebrate is a rodent, such as a mouse, rat, guinea pig or rabbit; a bird such as a turkey, chicken, chicken or other broilers; farm animals such as a cow, horse, pig, piglet or other freely moving farm animals; or a pet such as a dog or cat. 4. The method according to claim 1, where the vertebrate is a human. 5. The method according to any one of claims 1 to 4, where the amino acid is any essential amino acid. 6. The method according to claim 5, where the essential amino acid is isoleucine, leucine, lysine and proline. 7. A method of reducing plasma glucose absorption in a vertebrate animal, including a mammal and a bird, in which AKG, AKG mono- and dimetallic salts, chitosan-AKG, or mixtures thereof, are administered to a vertebrate, including a mammal and a bird, in an amount and / or frequency, sufficient to provide the desired effect on glucose absorption. 8. A method for preventing, inhibiting or alleviating a high plasma glucose condition in a vertebrate animal, including a mammal and a bird, in which AKG, AKG mono- and dimetallic salts, chitosan-AKG, or mixtures thereof, are administered to a vertebrate animal, including a mammal and a bird in an amount and / or with a frequency sufficient to provide the desired effect in relation to the specified condition. 9. The method according to any one of claims 7 and 8, wherein the mono- and dimetallic salts of AKG are selected from the group consisting of CaAKG, Ca (AKG) ₂ and NaAKG. 10. The method according to any one of claims 7 and 8, wherein the vertebrate is a rodent such as a mouse, rat, guinea pig or rabbit; a bird such as a turkey, chicken, chicken or other broilers; farm animals such as a cow, horse, pig, piglet or other freely moving farm animals; or a pet such as a dog or cat. 11. The method according to any one of claims 7 and 8, wherein the vertebrate is a human. 12. The method of claim 8, wherein the high plasma glucose condition is type I or type II diabetes mellitus. 13. The use of AKG, mono- and dimetallic salts of AKG, chitosan-AKG, or mixtures thereof, in a therapeutically effective amount for the manufacture of a composition for the prevention, alleviation or treatment of a condition with high plasma glucose. 14. The use of claim 13, wherein the high plasma glucose condition is type I or type II diabetes mellitus. 15. The use of AKG, mono- and dimetallic salts of AKG, chitosan-AKG or mixtures thereof in a therapeutically effective amount for the manufacture of a composition for improving absorption, altered absorption, poor absorption and impaired absorption of amino acids and / or peptides. 16. The use according to any one of claims 13 and 15, wherein the composition is a pharmaceutical composition optionally with a pharmaceutically acceptable carrier and / or additives. 17. The use according to any one of paragraphs.13 and 15, where the composition is a food or nutritional supplement. 18. The use of claim 17, wherein the food or food supplement is a dietary supplement and / or a component in the form of a solid food and / or drink. 19. The use according to any one of paragraphs.13 and 15, where the therapeutically effective amount is 0.01-0.2 g / kg of body weight per daily dose.

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